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Human- and Mouse-Inducible Nitric Oxide Synthase Promoters Require Activation of Phosphatidylcholine-Specific Phospholipase C and NF-κB

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Abstract

Background

The production of nitric oxide by type II inducible nitric oxide synthase (type II NOS) gene is controlled at least in part by transcriptional activation. Although the murine and human type II NOS genes share significant sequence homology, they differ in the induction stimuli required for activation.

Materials and Methods

The A549 human and murine RAW 264.7 cell lines were cultured in the presence of inducers of the type II NOS gene and exposed to specific inhibitors of phosphatidyl choline-specific phospholipase C, NF-κB, and endocytosis, as well as to reagents that deplete stores of ATP or prevent the acidification of endosomes. The effect of these reagents on the induction of the type II NOS gene transcription, translation, and NO expression was studied using electromobility shift assays. Western blotting, and the detection of NO as nitrates, as appropriate. Additionally, the ability of the native human type II NOS NF-κB recognition sequence to bind NF-κB was compared with a concensus sequence and with a mutated oligomer.

Results

Type II NOS production by both human and mouse cells could be prevented by the addition of the specific inhibitor of phosphatidylcholine-specific phospholipase C, D609, and of agents that interfere with the activation of NF-κB. Both mouse and human cells also required acidic endosome formation and the production of 1,2-diacylglycerol for type II NOS expression. Additionally, the native human type II NOS NF-κB recognition sequence bound NF-κB with significantly less affinity than did the recognition sequence derived from the human immunoglobulin light-chain gene promoter.

Conclusions

These experiments show that whereas mouse cells can be activated by lipopolysaccharide to produce nitric oxide, and human cells require activation by a mixture of cytokines to produce nitric oxide, the intracellular activation pathway following receptor binding of these heterologous stimuli is shared. Additionally, NF-κB activation is necessary but not sufficient for inducible nitric oxide synthase production in human cells, in contrast to murine cells in which it serves as a complete inducer.

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References

  1. Moncada S, Higgs A. (1993) The L-argininenitric oxide pathway. New Engl. J. Med. 329: 2002–2012.

    Article  CAS  PubMed  Google Scholar 

  2. Garthwaite J, Charles SL, Chess-Williams R. (1988) Endothelium-derived relaxing factor release on activation of NMDA receptors suggests a role as intercellular messenger in the brain. Nature 336: 385–388.

    Article  CAS  Google Scholar 

  3. Nathan CF, Hibbs JB, Jr. (1991) Role of nitric oxide synthesis in macrophage antimicrobial activity. Curr. Opin. Immunol. 3: 65–70.

    Article  CAS  Google Scholar 

  4. Xue C, Rengasamy A, Le Cras TD, Koberna PA, Dailey GC, Johns RA. (1994) Distribution of NOS in normoxic vs. hypoxic rat lung: Upregulation of NOS by chronic hypoxia. Am. J. Physiol. 267: L667–L678.

    PubMed  CAS  Google Scholar 

  5. Chen S, Aston-Jones G. (1994) Cerebellar injury induces NADPH diaphorase in Purkinje and inferior olivary neurons in the rat. Exp. Neurol 126: 270–276.

    Article  CAS  PubMed  Google Scholar 

  6. Mannick JB, Asano K, Izumi K, Kieff E, Stamler JS. (1994) Nitric oxide produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation. Cell 79: 1137–1146.

    Article  CAS  PubMed  Google Scholar 

  7. Nussler AK, Billiar TR. (1993) Inflammation, immunoregulation, and inducible nitric oxide synthase J. Leuk. Biol. 54: 171–178.

    Article  CAS  Google Scholar 

  8. Stuehr DJ, Marietta MA. (1985) Mammalian nitrate biosynthesis: Mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide. Proc. Natl. Acad. Sei. U.S.A. 82: 7738–7742.

    Article  CAS  Google Scholar 

  9. Lorsbach RB, Murphy WJ, Lowenstein CJ, Snyder SH, Russell SW. (1993) Expression of the nitric oxide synthase gene in mouse macrophages activated for tumor cell killing. Molecular basis for the synergy between interferon-gamma and lipopoly saccharide. J. Biol Chem. 268: 1908–1913.

    PubMed  CAS  Google Scholar 

  10. De Maria R, Graza Cifone M, Trotta R, Rippo MR, Festuccia C, Santoni A, Testi R. (1994) Triggering of human monocyte activation through CD69, a member of the natural killer cell gene complex family of signal transducing receptors. J. Exp. Med. 180: 1999–2004.

    Article  PubMed  Google Scholar 

  11. Bukrinsky MI, Nottet HSLM, Schmidtmayerova H, Dubrovsky L, Flanagan CR, Mullins ME, Lipton SA, Gendleman HE. (1995) Regulation of nitric oxide synthase activity in human immunodeficiency virus type 1 (HIV-1)-infected monocytes: Implications for HIV-associated neurological disease. J. Exp. Med. 181: 735–745.

    Article  CAS  PubMed  Google Scholar 

  12. Martin E, Nathan C, Xie Q-w. (1994) Role of interferon regulatory factor 1 in induction of nitric oxide synthase. J. Exp. Med. 180: 977–984.

    Article  CAS  Google Scholar 

  13. Xie Q-w, Kashiwabara Y, Nathan C. (1994) Role of transcription factor NF-kB/Rel in induction of nitric oxide synthase. J. Biol Chem. 269: 4705–4709.

    PubMed  CAS  Google Scholar 

  14. Spitsin SV, Koprowski H, Michaels FH. (1996) Characterization and functional analysis of the human inducible nitric oxide synthase gene promoter. Mol. Med. 2: 226–235.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. de Vera ME, Shapiro RA, Nussler AK, Mudgett JS, Simmons RL, Morris SM, Jr, BilliarTR, Geller DA. (1996) Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter. Proc. Natl. Acad. Sei. U.S.A. 93: 1054–1059.

    Article  Google Scholar 

  16. Kronke M, Schultze S, Scheurich P, Pfizenmaier K. (1992) TNF signal transduction and TNF responsive genes. In: Aggarwal, BB, Vilcek J (eds). Tumor Necrosis Factor: Structure, Function and Mechanism of Action. Marcel Dekker, New York, pp. 189–216.

    Google Scholar 

  17. Leung K, Betts JC, Xu L, Nabel GJ. (1994) The cytoplasmic domain of the interleukin-1 receptor is required for nuclear factor-kappa B signal transduction. J. Biol. Chem. 269: 1579–1582.

    PubMed  CAS  Google Scholar 

  18. Asano K, Chee CB, Gaston B, Lilly CM, Gerard C, Drazen JM, Stamler JS. (1994) Constitutive and inducible nitric oxide gene exression, regulation, and activity in humanlung epithelial cells. Proc. Natl. Acad. Sei. U.S.A. 91: 10089–10093.

    Article  CAS  Google Scholar 

  19. Ray KP, Kennard N. (1993) Interleukin-1 induces a nuclear form of transcription factor NF kappa B in human lung epithelial cells. Agents Actions Suppl. 38: C61–C63.

    Article  CAS  Google Scholar 

  20. O′Connor KJ, Moncada S. (1991) Glucocorticoids inhibit the induction of nitric oxide synthase and the related cell damage in adenocarcinoma cells. Biochim. Biophys. Acta 1097: 227–231.

    Article  PubMed  Google Scholar 

  21. Geller DA, Nussler AK, Di Silvio M, Lowenstein CJ, Shapiro RA, Wang SC, Simmons RL, Billiar TR. (1993) Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc. Natl. Acad. Sei. U.S.A. 90: 522–526.

    Article  CAS  Google Scholar 

  22. Scheinman RI, Cogswell PC, Lofquist AK, Baldwin AS, Jr. (1995) Role of transcriptional activation of IkBa in mediation of immunosuppression by glucocorticoids. Science 270: 283–286.

    Article  CAS  PubMed  Google Scholar 

  23. Auphan N, DiDonato JA, Rosette C, Helmberg A, Karin M. (1995) Immunosuppression by glucocorticoids: Inhibition of NFkB activity through induction of IκB synthesis. Science 270: 286–290.

    Article  CAS  PubMed  Google Scholar 

  24. Kleinert H, Euchenhofer C, Ihrig-Biedert I, Forstermann U. (1996) Glucocorticoids inhibit the induction of nitric oxide synthase II by down-regulating cytokine-induced activity of transcription factor nuclear factor-kappa B. Mol. Pharmacol 49: 15–21.

    PubMed  CAS  Google Scholar 

  25. Muller JM, Ziegler-Heitbrock JHW, Baeuerle PA. (1993) Nuclear factor κB, a mediator of lipopolysaccharide effects. Immunobiology 187: 233–256.

    Article  CAS  PubMed  Google Scholar 

  26. Weinstein SL, Gold MR, DeFranco AL. (1991) Bacterial lipopolysaccharide stimulates protein tyrosine phosphorylation in macrophages. Proc. Natl Acad. Sei. U.S.A. 88: 4148–4152.

    Article  CAS  Google Scholar 

  27. Moehren G, Gustavsson L, Hoek JB. (1994) Activation and desensitization of phospholipase D in intact rat hepatocytes. J. Biol. Chem. 269: 838–848.

    PubMed  CAS  Google Scholar 

  28. Stein B, Rahmsdorf HJ, Steffen A, Litfin M, Herrlich P. (1989) UV-induced DNA damage is an intermediate step in UV-induced expression of human immunodeficiency virus type 1, collagenase, c-fos, and metallothionein. Mol. Cell Biol. 9: 5169–5181.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Baeuerle PA, Henkel T. (1994) Function and activation of NF/κB in the immune system. Annu. Rev. Immunol 12: 141–179.

    Article  CAS  PubMed  Google Scholar 

  30. Schtuze S, Potthoff K, Machliedt T, Berkovic D, Weigmann K, Kronke M. (1992) TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced “acidic” sphingomyelin breakdown. Cell 71: 765–776.

    Article  Google Scholar 

  31. Xie Q-W, Whisnant R, Nathan C. (1993) Promoter of the mouse gene promoting calcium-independent nitric oxide synthase confers inducibility by interferon gamma and lipopolysaccharide. J. Exp. Med. 177: 1779–1784.

    Article  CAS  Google Scholar 

  32. Nunokawa Y, Ishida N, Tanaka S. (1994) Promoter analysis of human inducible nitric oxide synthase gene associated with cardiovascular homeostasis. Biochem. Biophys. Res. Commun. 200: 802–807.

    Article  CAS  Google Scholar 

  33. Chartrain NA, Geller DA, Koty PP, Sitrin NF, Nussler AK, Hoffman EP, Billiar TR, Hutchinson NI, Mudgett JS. (1994) Molecular cloning, structure, and chromosomal localization of the human inducible nitric oxide synthase gene. J. Biol. Chem. 269: 6765–6772.

    PubMed  CAS  Google Scholar 

  34. Muller-Decker K. (1989) Interruption of TPA-induced signals by an antiviral and antitumoral xanthate compound: Inhibition of phospholipase C-type reaction. Biochem. Biophys. Res. Commun. 162: 198–205.

    Article  CAS  PubMed  Google Scholar 

  35. Kuno K, Matsushima K. (1994) The IL-1 receptor signaling pathway. J. Leuk. Biol. 56: 542–547.

    Article  CAS  Google Scholar 

  36. Kracht M, Heiner A, Resch K, Szamel M. (1993) Interleukin-1 induced signalling in T cells. J. Biol. Chem. 268: 21066–21072.

    PubMed  CAS  Google Scholar 

  37. Kolesnick R, Golde DW. (1994) The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling. Cell 77: 325–328.

    Article  CAS  PubMed  Google Scholar 

  38. Weigmann K, Schutze S, Machliedt T, Witte D, Kronke M. (1994) Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 78: 1005–1015.

    Article  Google Scholar 

  39. Mellman I, Fuchs R, Helenius A. (1986) Acidification of the endocytic and exocytic pathways. 55: 663–700.

    CAS  Google Scholar 

  40. Robinson PJ. (1991) Phosphatidylinositol membrane anchors and T cell activation. Immunol Today 12: 35–41.

    Article  CAS  PubMed  Google Scholar 

  41. Ulevitch RJ, Tobias PS. (1995) Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu. Rev. Immunol 13: 437–457.

    Article  CAS  PubMed  Google Scholar 

  42. Mathias S, Younes A, Kan CC, Orlow I, Joseph C, Kolesnick RN. (1993) Activation of the sphingomyelin signaling pathway in intact EL-4 cells and in a cell-free system by IL-lb. Science 259: 519–522.

    Article  CAS  PubMed  Google Scholar 

  43. Dobrowsky RT, Hannun YA. (1992) Ceramide stimulates a cytosolic protein phosphatase. J. Biol. Chem. 267: 5048–5051.

    PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by the Commonwealth of Pennsylvania.

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Authors and Affiliations

  1. Biotechnology Foundation Laboratories, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

    Sergei V. Spitsin, Michael Bertovich, Hilary Koprowski & Frank H. Michaels

  2. Department of Pathology, Thomas Jefferson University, Room 462, Jefferson Alumni Hall, 1020 Locust Street, Philadelphia, Pennsylvania, 19107, USA

    John L. Farber & Gisela Moehren

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  1. Sergei V. Spitsin
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  2. John L. Farber
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Communicated by A. Levine.

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Spitsin, S.V., Farber, J.L., Bertovich, M. et al. Human- and Mouse-Inducible Nitric Oxide Synthase Promoters Require Activation of Phosphatidylcholine-Specific Phospholipase C and NF-κB. Mol Med 3, 315–326 (1997). https://doi.org/10.1007/BF03401810

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